The herpes viruses include the herpes simplex viruses, comprising two closely related variants designated types 1 (HSV-1) and 2 (HSV-2). These types cross react strongly, but can be distinguished by neutralization titrations. HSV-1 and HSV-2 are responsible for a variety of human diseases, such as skin infection, genital herpes, viral encephalitis, and the like.
The herpes simplex virus is a double stranded DNA virus having a genome of about 150 to 160 Kb, packaged within an icosahedral nucleocapsid, enveloped in a membrane. The membrane includes a number of virus-specific glycoproteins, the most abundant of which are gB, gC, gD, and gE. The proteins gB and gD are cross-reactive between HSV-1 and HSV-2.
It is a matter of great medical and scientific interest to provide safe and effective vaccines for humans against both HSV-1 and HSV-2, and where infection has occurred, to provide therapies for treatment of the disease. One promising approach has been the use of isolated glycoproteins, which have been shown to provide protection when injected into mice subsequently challenged with live virus. These proteins are produced by, e.g., the methods taught in U.S. Pat. No. 4,618,578, incorporated herein by reference.
Another critical aspect affecting vaccine efficacy is the vehicle employed to deliver the antigen. Proper activation of the immune system depends on a complex series of interactions between the immune system's various components, which include antigen presenting cells (APC), e.g., macrophages, dermal Langerhans cells, and dendritic cells; helper thymocytes (T.sub.h -cells); cytotoxic thymocytes (T.sub.c -cells); and immunoglobulin-secreting B-cells. APCs ingest foreign particles in the body, partially digest the antigens thereon, and "present" the partially-digested antigens on their surfaces in association with proteins encoded in the major histocompatability complex (MHC) genes. MHC proteins are classified as type I (which are expressed on the surfaces of all nucleated cells), and type II, which are expressed only on certain cells of the immune system. Type II proteins are interchangeably denoted as MHC Ia (for "immune-associated"). APCs express both MHC I and Ia, but it is the Ia-antigen complexes that are important for initial activition of the immune system. These antigen-MHC Ia complexes are recognized by T.sub.h cells that bear a T-cell receptor capable of binding to the complex. This binding, in addition to the secretion of certain cytokines (particularly interleukin-1, "IL-1"), effects activation of the T.sub.h cell. As a result, the stimulated T.sub.h cells proliferate and begin secreting other cytokines, eventually activating T.sub.c cells and B-cells specific for the antigen that originally started the cascade. T.sub.c cells recognize antigen in combination with MHC I proteins on cell surfaces, and effect lysis of such cells in a process known as cell mediated immunity (CMI) Thus, T.sub.c cells and CMI are believed to be particularly important in the immune response to viral infection, as lysis of cells exhibiting viral antigens results in interruption of viral reproduction.
B-cells express antibodies of the various classes in a process known as humoral immunity. The antibody classes most important in resistance to infection are IgM, IgG, and IgA. Antibodies may also play a role in protection from viral infection, by fixing complement on infected cells, by clearing viral particles from the bloodstream, by opsonizing cells for clearance by macrophages and neutrophils, and by initiating antibody-dependent cell-mediated immunity (ADCC), which is effected by killer cells (K).
The efficacy of these immune defenses varies depending upon the pathogen's antigenicity and mode of infection. When designing a vaccine, the object is to provide for activation of those immune defenses which will result in the most effective protection from the pathogen in question. In general, an adjuvant is often employed in order to increase the immune response to a particular antigen. Most adjuvants, however, are only able to enhance humoral immunity, and fail to increase cell mediated immunity.
The mode of administration is also of critical importance. Most vaccines are administered intramuscularly or subcutaneously (in rare cases intradermally), as lymph in these tissues drains into the lymph nodes where the antigens will be accessible to T.sub.h cells and macrophages. In contrast, administration of soluble antigens by intravenous routes may result in tolerance to the antigen, i.e., a suppression of any immune response.
The archetypal adjuvant is Freund's adjuvant, which is commonly used in laboratory immunology. Freund's complete adjuvant (FCA) consists of an oil-in-water emulsion of mineral oil, surfactant, and killed mycobacteria (M. tuberculosis). Freund's incomplete adjuvant (FIA) omits the mycobacteria. These adjuvants provide an enhanced immune response, but are not suitable for administration to humans, as mycobacteria are the causative agent of tuberculosis, and may cause adverse responses detrimental to the subject. Further, the mineral oil component is not metabolized, and may cause abcesses and granulomas at the site of injection. Researchers have since replaced the mycobacteria with muramyl dipeptide (MDP) derivatives, which were determined to be the smallest component of the mycobacterial cell walls that retained the adjuvant activity. The preparation of various MDP derivatives and analogs is described in detail in U.S. Pat. Nos. 4,082,735; 4,082,736; 4,101,536; 4,185,089; 4,235,771; and 4,406,890. It is currently believed that the MDP derivative stimulates the release of IL-1. The mineral oil component has been replaced by various synthetic and metabolizable vegetable oils. See for example Allison et al, U.S. Pat. No. 4,606,918, disclosing an adjuvant formulation comprising muramyl dipeptide, a polyoxypropylene-polyoxyethylene block polymer (Pluronic.RTM.), a non-ionic surfactant, and a metabolizable oil such as squalane. Other adjuvants known in the art include alum, lipid A, trehalose dimycolate, and dimethyldioctadecylammonium bromide (DDA). However, only alum has been approved for human administration.
Liposomes have also been employed to deliver biologically active material. See for example Allison, U.S. Pat. No. 4,053,585, which discloses the administration of several antigens in negatively-charged liposomes, optionally including killed M. tuberculosis. Fullerton et al, U.S. Pat. No. 4,261,975, discloses the use of separated influenza membranes, with hemagglutinin spikes attached, which is bound to liposomes for use in influenza vaccines. Tarcsay et al, U.S. Pat. No. 4,406,890, discloses the use of liposomes to encapsulate MTP-PE (a lipophilic MDP derivative) and bovine serum albumin (a standard model antigen). Liposomes are small vesicles composed of amphipathic lipids arranged in bilayers. Liposomes are usually classified as small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), or multi-lamellar vesicles (MLV). SUVs and LUVs have only one bilayer, whereas MLVs contain many concentric bilayers. Liposomes may be used to encapsulate various materials, by trapping hydrophilic compounds in the interior or between bilayers, or by trapping hydrophobic compounds in the bilayer.
Liposomes exhibit a wide variety of characteristics, depending upon their size, composition, and charge. For example, liposomes having a small percentage of unsaturated lipids tend to be slightly more permeable, while liposomes incorporating cholesterol tend to be more rigid and less permeable. Liposomes may be positive, negative, or neutral in charge, depending on the hydrophilic group. For example, choline-based lipids impart a positive charge, phosphate and sulfate based lipids contribute a negative charge, and glycerol-based lipids tend to be neutral in solution.
Several HSV vaccines have been prepared. S. Dundarov et al, Dev Biol Standard, 52:351-57 (1982) disclosed the treatment of humans with formalin-inactivated HSV in distilled water. GRB Skinner et al, Dev Biol Standard, 52:333-44 (1982) disclosed the treatment of humans with formalin-inactivated HSV in saline. L. Chan, Immunol, 49:343-52 (1983) disclosed the protective immunization of mice against HSV challenge by vaccination with gD in saline. Kino et al, U.S. Pat. No. 4,661,349 disclosed vaccines comprising purified HSV gB with alum. Person, U.S. Pat. No. 4,642,333 disclosed HSV gB and its administration to rabbits in Freund's adjuvant. Recently, L.R. Stanberry et al, J Infect Dis, 157:156-163 (1988) reported the use of rgD and rgB in a vaccine to ameliorate the symptoms of genital herpes infection in guinea pigs.